This invention relates to pharmaceutical compositions, particularly liposomal oligonucleotide compositions, their preparation and their use.
In WO 95/02069 there are described oligonucleotides specifically hybridizable with DNA or RNA derived from a protein kinase C (PKC) gene, which oligonucleotides are particularly for use in the diagnosis and treatment of neoplastic, hyperproliferative and inflammatory disorders associated with protein kinase C.
It has now been found that compositions retaining high activity after prolonged circulation in the bloodstream and exhibiting reduced accumulation of oligonucleotide in non-target organs such as the liver and kidney can be prepared by formulation of such oligonucleotides within sterically stabilised liposomes.
Accordingly, the present invention provides a pharmaceutical composition comprising (A) an oligonucleotide having 5 to 50 nucleotide units specifically hybridizable with DNA or RNA derived from a protein kinase C gene, entrapped in (B) sterically stabilised liposomes.
Hybridisation, in the context of nucleic acid chemistry, means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them. xe2x80x9cSpecifically hybridizablexe2x80x9d and xe2x80x9ccomplementaryxe2x80x9d are terms which are used to indicate a sufficient degree of complementary such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable.
An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementary to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment, or, in the case of in vitro assays, under conditions in which the assays are conducted.
As used in the context of this invention, the term xe2x80x9coligonucleotidexe2x80x9d refers to a substance having a plurality of nucleotide units formed from naturally occurring bases and sugars joined by phosphodiester internucleoside (backbone) linkages. The term xe2x80x9coligonucleotidexe2x80x9d also includes analogues which function similarly to naturally occurring oligonucleotides but which have non-naturally occurring monomers (nucleotides) or portions thereof. These oligonucleotide analogues are often preferred over native forms because of properties such as enhanced cellular uptake, enhanced target binding affinity and increased stability in the presence of nucleases.
In preferred embodiments of the invention, the oligonucleotide (A) is specifically hybridizable with the translation initiation codon of the PKC gene, in which case it preferably comprises a CAT sequence, or with the 5xe2x80x2 untranslated region or 3xe2x80x2 untranslated region of the gene. In other preferred embodiments of the invention, the oligonucleotide (A) is specifically hybridizable with DNA or mRNA encoding a particular PKC isozyme (isoform) or a particular set of PKC isozymes.
The oligonucleotide (A) preferably comprises from 8 to 30 nucleotide units, more preferably 12 to 25 nucleotide units, especially 18 to 22 nucleotide units.
In some preferred oligonucleotides (A), at least one nucleotide is modified at the 2xe2x80x2 position of the sugar moiety. Certain preferred oligonucleotides (A) are chimeric oligonucleotides. xe2x80x9cChimeric oligonucleotidesxe2x80x9d or xe2x80x9cchimerasxe2x80x9d, in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the RNA target) and a region that is a substrate for RNase H cleavage. In one preferred embodiment, a chimeric oligonucleotide comprises at least one region modified to increase target binding affinity and, usually, a region that acts as a substrate for RNAse H. Affinity of an oligonucleotide for its target is routinely determined by measuring the Tm of an oligonucleotide/target pair, which is the temperature at which the oligonucleotide and target dissociate; dissociation is detected spectrophotometrically. The higher the Tm, the greater the affinity of the oligonucleotide for the target. In a more preferred embodiment, the region of the oligonucleotide which is modified to increase target binding affinity comprises at least one nucleotide modified at the 2xe2x80x2 position of the sugar, particularly a 2xe2x80x2-alkoxy, 2xe2x80x2-alkoxyalkoxy or 2xe2x80x2-fluoro-modified nucleotide. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2xe2x80x2-deoxyoligonucleotides against a given target. In the chimeric oligonucleotides, the region which is a substrate for RNAse H comprises at least one 2xe2x80x2-deoxynucleotide. RNAse H is a cellular endonuclease that cleaves the RNA strand of RNA:DNA duplexes; activation of this enzyme therefore results in cleavage of the RNA target, and thus can greatly enhance the efficiency of antisense inhibition. Cleavage of the RNA target can be routinely demonstrated by gel electrophoresis. In another preferred embodiment, the chimeric oligonucleotide is also modified to enhance nuclease resistance. Cells contain a variety of exo- and endo-nucleases which can degrade nucleic acids. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide. Nuclease resistance is routinely measured by incubating oligonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oligonucleotides which have been modified to enhance their nuclease resistance survive intact for a longer time than unmodified oligonucleotides. A variety of oligonucleotide modifications have been demonstrated to enhance or confer nuclease resistance. Oligonucleotides which contain at least one phosphorothioate modification are presently more preferred. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease resistance.
Specific examples of some preferred oligonucleotides may contain phosphorothioate, phosphotriester, methyl phosphonate, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar (xe2x80x9cbackbonexe2x80x9d) linkages, e.g. amide-type linkages. Most preferred are phosphorothioates and those with CH2xe2x80x94NHxe2x80x94Oxe2x80x94CH2, CH2xe2x80x94N(CH3)xe2x80x94Oxe2x80x94CH2, CH2xe2x80x94Oxe2x80x94N(CH3)xe2x80x94CH2, CH2xe2x80x94N(CH3)xe2x80x94N(CH3)xe2x80x94CH2 and Oxe2x80x94N(CH3)xe2x80x94CH2xe2x80x94CH2 and CH2xe2x80x94C(O)xe2x80x94NHxe2x80x94CH2 backbones (where phosphodiester is Oxe2x80x94Pxe2x80x94Oxe2x80x94CH2). Also preferred are oligonucleotides having morpholino backbone structures, for example as described in U.S. Pat. No. 5,034,506. In other preferred embodiments, such as the protein-nucleic acid or peptide-nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, as described by P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science 1991, 254, 1497. Other preferred oligonucleotides may contain sugar moieties comprising one of the following at the 2xe2x80x2 position: OH; SH; SCH3; F; OCN; OCH2OCH3; O(CH2CH2)mOCH3 wherein m is 1, 2 or 3, preferably OCH2CH2OCH3; OCH2O(CH2)nCH3; O(CH2)nNH2 or O(CH2)nCH3 where n is from 1 to about 10; OCH2CH2NR1R2 wherein R1 and R2 are independently of each other, H or CH3. C1 to C10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; C1-10 lower alkoxy or substituted alkoxy, preferably OCH3; Cl; Br; CN; CF3; OCF3; Oxe2x80x94, Sxe2x80x94, or N-alkyl; Oxe2x80x94, Sxe2x80x94, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a cholesteryl group; a conjugate; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group. Other preferred embodiments may include at least one modified base form or xe2x80x9cuniversal basexe2x80x9d such as inosine. Preferred bases includexanthine, hypoxanthine, adenine, 2-aminoadenine, guanine, 6-thioguanine, uracil, thymine, cytosine, 5-methylcytosine, 5-propynyluracil, 5-fluorouracil and 5-propynylcytosine.
In certain especially preferred embodiments of the invention, all nucleotides of the oligonucleotide (A) are 2xe2x80x2-deoxynucleotides and all backbone linkages are phosphorothioate linkages.
In certain other especially preferred embodiments, the oligonucleotide (A) is a chimeric oligonucleotide having one or more regions with 2xe2x80x2-deoxynucleotides and one or more regions with 2xe2x80x2-modified nucleotides, preferably 2xe2x80x2-alkoxynucleotides or 2xe2x80x2-alkoxyalkoxynucleotides, particularly 2xe2x80x2-methoxyethoxynucleotides, the one or more 2xe2x80x2-deoxynucleotide regions preferably having phosphorothioate backbone linkages and the one or more 2xe2x80x2-modified nucleotide regions preferably having phosphodiester or phosphorothioate backbone linkages. These chimeric oligonucleotides preferably comprise a region of 2xe2x80x2-deoxynucleotides between two regions of 2xe2x80x2-modified nucleotides, the deoxynucleotide region being preferably at least 4 nucleotides long, more preferably at least 6 nucleotides long, especially at least 8 nucleotides long.
The oligonucleotides used as component (A) of the composition of the invention may be conveniently and routinely made using well-known techniques such as solid phase synthesis. Equipment for such synthesis is available commercially from various sources including Applied Biosystems. The use of such techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives is well known. It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and/or CPG (available from Glen Research, Sterling Va.) to synthesize fluorescently labelled, biotinylated or other modified oligonucleotides such as cholesterol-modified oligonucleotides.
Specific especially preferred oligonucleotides, for which the nucleotide sequences and preparation have been published in WO95/02069, include the following:
Most preferred among the oligonucleotides hereinbefore described are those having SEQUENCE ID No. 2,3 or 5.
In compositions of the invention, the oligonucleotide (A) is entrapped in sterically stablised liposomes (B). Examples of sterically stabilised liposomes are those in which part of the lipid is a glycolipid, particularly ganglioside GM, saturated phosphatidylinositol or galactocerebroside sulphate ester, such as those described in WO 88/04924; those in which part of the lipid is derivatised with hydrophilic polymer such as those described in WO 91/05545 or U.S. Pat. No. 5,225,212; and those comprising a vesicle-forming lipid and a lipid-polymer conjugate having a hydrophobic moiety and a polar head group, such as those described in WO 94/20073.
In a preferred embodiment of the invention, the liposomes (B) comprise at least one underivatised vesicle-forming lipid and at least one vesicle-forming lipid derivatised with hydrophilic polymer which may be, for example, a polymer containing a hydroxy and/or carboxyl group such as a polylactic acid, a polyglycolic acid or, preferably, a polyethylene-glycol. More preferably, the hydrophilic polymer is a polyethyleneglycol having a molecular weight of 1000 to 5000 daltons, such as 1500 to 2500 daltons, especially 1800 to 2200 daltons. The hydrophilic polymer is preferably derivatised with a polar head group of a phospholipid, especially a phospholipid having an amino head group, i.e. the derivatised lipid is preferably a phospholipid having an amino group, especially a phosphatidylethanolamine such as dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine or, particularly, distearoyl phosphatidylethanolamine.
Various methods of derivatising an amino-containing lipid with a hydroxyl- and/or carboxyl-containing hydrophilic polymer will be apparent to those skilled in the art. Several such methods are described in WO 91/05545 and U.S. Pat. No. 5,225,212; the phospholipid having an amino group may be derivatised with the hydrophilic polymer by any of these methods. Preferably, the phospholipid having an amino group is derivatised with a hydroxyl-containing hydrophilic polymer such that the polymer is attached to the phospholipid through a carbamate linkage; this may be achieved by reacting a hydroxyl group of the polymer (other hydroxyl groups being capped, if necessary in view of their reactivity, for example by etherification) with diimidazole to give an activated imidazolexe2x80x94terminated polymer which is then reacted with the amino-containing phospholipid to couple the phospholipid to the hydrophilic polymer through a carbamate group, as described in WO 91/05545 or U.S. Pat. No. 5,225,212. In an especially preferred embodiment of the invention, the derivatised lipid is an amino-containing phospholipid, particularly a phosphatidylethanolamine, coupled through a carbamate group to a polyethyleneglycol capped at one end by an alkoxy group, particularly a methoxy or ethoxy group. Such a derivatised lipid is available commercially.
The derivatised lipid is generally present in a minor molar amount relative to the total lipid content of the liposomes, preferably in an amount of 1 to 20 mole % of the total lipid content, although a lower amount, for example 0.1 mole %, may be appropriate when the derivatised lipid has a high molecular weight. The major part of the lipid content of the liposomes generally comprises one or more underivatised vesicle-forming lipids such as are used in conventional liposomes. Such lipids include, for example, lipids having two hydrocarbon chains, usually in acyl groups, and a polar head group, including phospholipids, for example phosphatidylcholines such as dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dilinoleoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, phosphatidylethanolamines such as those mentioned hereinbefore, and phosphatidic acids such as dimyristoyl phosphatidic acid and dipalmitoyl phosphatidic acid. Other conventionally used lipids include sterols, particularly cholesterol, and glycolipids such as those mentioned hereinbefore. Preferably, the underivatised lipid comprises a mixture of a phospholipid, especially a phosphatidylcholine, and a sterol, especially cholesterol.
In the abovementioned preferred embodiment, the sterically stabilised liposomes (B) preferably comprise 4-10 mol % of the derivatised lipid, 40-80 mol % of the underivatised phospholipid and 20-50 mol % of the sterol. In especially preferred liposomes (B), the molar ratio of derivatised lipid: underivatised phospholipid: sterol is 1:10:5.
In another preferred embodiment of the invention, the liposomes (B) comprise (i) a glycolipid together with (ii) a vesicle-forming phospholipid or sphingolipid or mixture thereof and, optionally, (iii) a sterol and/or an acylglycerol lipid. The glycolipid is preferably a negatively charged glycolipid, especially ganglioside GM1 (monosialoganglioside) or hydrogenated phosphatidylinositol. The vesicle-forming phospholipid may be one or more of the phospholipids hereinbefore mentioned, preferably a phosphatidylcholine, a phosphatidylethanolamine or a mixture thereof. Especially preferred phospholipids are distearoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine. The sphingolipid is preferably sphingomyelin and is preferably used together with a phospholipid. The sterol may be, for example, ergosterol or, preferably, cholesterol. The acylglycerol lipid may be an ester of glycerol containing two fatty acid acyl groups each having at least 12 carbon atoms, for example lauroyl, myristoyl, palmitoyl or oleoyl groups, and one acyl group of formula R1COxe2x80x94, where R1 is a residue, containing up to 10 carbon atoms, of a monocarboxylic acid of formula R1COOH after removal of the xe2x80x94COOH group or, preferably, of formula xe2x80x94COR2COOH where R2 is a residue, containing up to 10 carbon atoms, preferably 1 to 4 carbon atoms, of a dicarboxylic acid of formula HOOCxe2x80x94R2xe2x80x94COOH, especially succinic acid, after removal of both xe2x80x94COOH groups. An especially preferred acylglycerol is 1,2-dipalmitoyl-sn-3-succinylglycerol.
In this second preferred embodiment of the invention, the liposomes preferably comprise (i) a negatively charged glycolipid together with (ii) a vesicle-forming phospholipid and/or sphingolipid and (iii) a sterol or acylglycerol lipid, especially (i) ganglioside GM1 or hydrogenated phosphatidylinositol together with (ii) distearoyl phosphatidylcholine or dioleoyl phosphatidylethanolamine or a mixture thereof with sphingomyelin and (iii) cholesterol or 1,2-dipalmitoyl-sn-3-succinylglycerol.
The liposomes may comprise from 2 to 20 mol % of the glycolipid (i) and 80 to 98 mol % of (ii) the phospholipid, sphingolipid or mixture thereof. In preferred embodiments, where the liposomes also comprise a sterol or acylglycerol, they may comprise 2 to 20 mol %, preferably 4 to 10 mol %, of the glycolipid, 40 to 80 mol %, preferably 60 to 80 mol %, of the phospholipid, sphingolipid or mixture thereof and 10 to 50 mol %, preferably 20 to 40 mol %, of the sterol or 5 to 40 mol %, preferably 10 to 30 mol %, of the acylglycerol.
Specific especially preferred liposomes (B) are those described hereinafter in the Examples.
The oligonucleotide-containing liposomes of the invention can be prepared using known methods for the preparation of drug-containing liposomes. For example, in one method, the lipid composition is dissolved in an organic solvent, such as an alcohol, ether, halohydrocarbon or mixture thereof, the solvent is removed from the resulting solution, for example by rotary evaporation or freeze drying, and the resulting lipid film is hydrated by dispersing in an aqueous medium, such as phosphate-buffered saline or an aqueous solution of a sugar, e.g. lactose, which medium also contains the oligonucleotide (A), to give an aqueous suspension of liposomes in the form of multilamellar vesicles (MLV""s). The aqueous liposome suspension may be treated to reduce the liposome size, for example to give small unilamellar vesicles (SUV""s), using known methods, for example by sonication or by extrusion through one or more membranes, e.g. polycarbonate membranes, having a selected pore size. Liposomes according to the invention preferably have on average a particle size below 500 nm, more preferably 50 to 200 nm, especially 80 to 120 nm.
It is generally desirable to have as high a weight ratio of oligonucleotide to lipid as possible consistent with liposome stability. The maximum for this weight ratio may vary depending on the nature and composition of the lipid component, but in general this maximum is likely to be about 1:20. Ratios between 1:40 and 1:400 can be used with good results.
The invention includes a method of modulating the expression of protein kinase C in cells which comprises contacting the cells with a composition of the invention as hereinbefore defined. The invention also includes a method of treating a condition associated with expression of protein kinase C which comprises administering a composition of the invention as hereinbefore defined to a mammal, particularly a human, or cells thereof, in need of such treatment.
The composition of the invention may be administered by pulmonary delivery or, preferably, parenterally, for example intravenously, subcutaneously, intraperitoneally or intramuscularly. Conditions which may be treated with the composition include hyperproliferative disorders such as psoriasis and mammalian cancer, particularly human cancer such as lung cancer, breast cancer, colorectal cancer and skin cancer.
For these indications, the appropriate dosage will, of course, vary depending upon the method of administration and on the severity and responsiveness of the condition to be treated. Individual doses and the administration regime can best be determined by individual judgement of a particular case of illness. However, in general, satisfactory results in animals are indicated to be obtained at daily dosages from about 0.6 mg/kg to about 6 mg/kg.
In larger mammals, for example humans, an indicated daily dose in the range of about 4.2 mg to about 420 mg conveniently administered, for example in divided doses of up to 3 times per day.